The primary objectives of this project are to provide greater insight into the control of eDNA generation (through autolysis) and processing (via staphylococcal nuclease) within a biofilm. These studies will focus on the varied micro-niches that exist within a staphylococcal biofilm, and define the metabolic and stoichiometric factors that influence the expression of genes involved in these processes during biofilm development. The proposed studies will extend our preliminary results testing the hypothesis that S. aureus biofilm produces distinct functional subpopulations in response to environmental and stochastic effects on gene expression. In testing this hypothesis we will establish and elucidate the functional roles of different functional subpopulations within a mature biofilm. To achieve these goals, we will perform three specific aims. The first aim will utilize a variety of transcriptional and metabolic probes, in combination with BioFlux microfluidics technology, to investigate the metabolic heterogeneity that arises during biofilm development and its impact on death and lysis. The second aim will study the regulation of nuclease expression during biofilm development, focusing heavily on the role of the Sae regulatory system, and the novel hypothesis that SaeP is a sensor of eDNA. The third aim will establish a division of labor within a biofilm and define the functional roles of the different subpopulations within a biofilm, including dispersal, mutagenesis, and antibiotic tolerance. Overall, the experiments described in these specific aims will rely on a highly collaborative effort to yield greater insight into the environmental and stochastic regulatory mechanisms that dictate the metabolism of different biofilm niches. In addition to providing a more complete understanding of the metabolic processes inherent to staphylococcal biofilm, this project will foster a burgeoning perspective of bacterial biofilm as a highly complex population of differentiated cells, akin to multicellular organisms.